Mesenchymal multi-potent stromal/stem cells (MSCs) have historically been characterized as stromal progenitors that can differentiate into a variety of cell types including bone, cartilage, muscle and fat. In recent years, elegant genetic and molecular studies have allowed enrichment of MSCs with higher progenitor potential. In mice, among the cell surface markers used for enrichment, co-expression of PDGFR?/ CD51 results in high enrichment of stem/progenitor activity. Likewise, LepR-Cre marks a long-term, self-renewing cell population with the highest progenitor potential compared to other Cres. During our studies of Hox11 function in the zeugopod skeleton (radius and ulna), we have made the surprising discovery that Hox11 protein expression is excluded from all differentiated cell types of the zeugopod, but is found uniquely in cells that co-express PDGFR?/CD51 and with cells that retain the LepR-Cre lineage trace. While not all PDGFR?+/CD51+ or LepRCre+ cells are Hox11+, all Hox11+ cells are PDGFR?/+CD51+ and LepRCre+. When comparing the in vitro CFU-F progenitor potential of PDGFR?/+CD51+/Hoxa11eGFP+ cells to the population double-positive for PDGFR?/+CD51+, we find that the PDGFR?/+CD51+/Hoxa11eGFP+ cells have three times higher progenitor potential than the double-positive total population, consistent with enriching for higher progenitor potential. Further, triple- positive cells can differentiate into cartilage, bone and adipocytes in vitro, further supporting their MSC potential. Unlike the commonly used markers for MSCs, Hox11 genes also function in these cells. Loss of Hox11 function eliminates the chondrogenic and osteogenic potential in this regionally restricted set of MSCs in vitro. Postnatal defects arising in Hox11 compound mutants as well as impaired fracture repair responses provide in vivo support for a continued role for Hox11 in MCSs throughout the life of the animal. Importantly, defects are only observed in the zeugopod; other skeletal compartments of Hox11 mutants exhibit normal MSC tri-lineage differentiation in vitro and are able to heal normally after fracture. In this application, we will use our Hox null mutants and Hoxa11eGFP reporter, in addition to a newly generated a Hoxa11-CreERT2 and Hoxd11 conditional allele that will allow us to prove in vivo lineage analyses, self-renewal potential and origin of this population, and dissection of continued function of this MSC population through skeletal development, postnatal growth, maintenance and repair. We will also explore preliminary data that shows that ALL endochondral bones maintain differential regional Hox expression in the bone marrow MSCs and investigate global function for Hox genes in regionally restricted MSC populations. Interrogation of these questions will directly impact our knowledge of skeletal development and bone biology, but also have important implications for the isolation and use of MSCs in tissue engineering and regenerative medicine approaches.